Radiolabelled Phenylethyl Imidazole Carboxylic Acid Ester Derivatives

ABSTRACT

Compounds derived from phenylethyl imidazole carboxylic acid esters have shown selective accumulation of radioactivity in the adrenal cortex, when labeled with a radioactive halogen. In particular, these compounds bind selectively to adrenocortical tissue facilitating the diagnosis of adrenal cortical masses such as incidentaloma, adenoma, primary and metastatic cortical carcinoma. Trace amounts are injected intravenously and accumulate rapidly in the adrenals, maintaining a high radioactivity plateau, which permits external imaging using computed SPECT (single photon emission) or PET (positron emission) techniques. 
     Independent of the position and type of the radioactive label, the compounds according to the invention are potent inhibitors of steroid P450c11 hydroxylation and bind with high affinity on sites of cortisol secretion. In order to avoid saturation of receptor sites, high specific activity labeling is mandatory for application in patients. The compounds in accordance with the invention have been found to possess an almost 1000-fold higher affinity when compared with the known, clinically used inhibitors (metyrapone, ketoconazole).

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part application of copending patentapplication Ser. No. 11/582,073, filed Oct. 17, 2006, which was adivisional of patent application Ser. No. 10/635,294, filed Aug. 6,2003, now U.S. Pat. No. 7,189,859 B2; the prior applications areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION Field of the Invention

Adrenal Scintigraphy for Detection of Adrenal Cortical Pathology.

BRIEF SUMMARY OF THE INVENTION

The invention relates to previously disclosed radioactively labelledderivatives of (R)-1-(1-phenylethyl)-1H-imidazole-5-carboxylic acidesters and methods for preparing these compounds. The invention alsorelates to the use of these radioactively labelled compounds asradiopharmaceuticals for functional diagnosis of adrenal disease and fortherapeutic applications. In particular, these compounds bindselectively to adrenocortical tissue facilitating the diagnosis ofadrenal cortical masses such as incidentaloma, adenoma, primary andmetastatic cortical carcinoma.

The present invention relates to a class of substituted(R)-1-(1-phenylethyl)-1H-imidazole-5-carboxylic acid esters, whichinteract selectively with the mitochondrial cytochrome P-450 species inthe adrenal cortex (Vanden Bossche, 1984). When labelled with aradiohalogen (iodine-123; bromine-76; fluorine-18, and others), thesecompounds serve as radiotracers for the diagnosis of adrenal corticalmasses such as incidentaloma, adenoma, primary and metastatic corticalcarcinoma. When labelled with a beta-emitting radionuclide (iodine-131;bromine-82, and others), these radiotracers may be used for radionuclidetherapy.

In particular, the compounds according to this invention are potentinhibitors of steroid P450c11 hydroxylation and bind with high affinityto sites of hormone production. In fact, the compounds in accordancewith this invention have been found to possess an almost 1000-foldselective affinity when compared with known, clinically used inhibitors(metyrapone, ketoconazole). Therefore, when injected intravenously, thelabelled derivatives of the present invention accumulate almostexclusively in the adrenals, reaching radioactivity levels that arediagnostically useful.

The parent compound etomidate (ethyl ester; ETO) is clinically used as ashort-acting hypnotic drug. When incubated with human adrenocorticaltissue slices, ETO was shown to block the conversion of 11-deoxycortisolto cortisol and of 11-deoxycorticosterone (DOC) to corticosterone andaldosterone (Weber 1993; Engelhardt 1994). Metomidate (MTO), the methylester, is an equally potent inhibitor of steroid 11β-hydroxylation.(R)-configuration of the methyl substituent at the chiral C-atom isessential for enzyme inhibition (Vanden Bossche, 1984; Berger et al.,2002; Zolle et al., 2008).

Clinical findings with [O-methyl-¹¹C]metomidate have indicated highuptake in lesions of adrenocortical origin, including adenomas, but verylow uptake in lesions of non-adrenocortical origin (Bergström 1998;2000). Asymptomatic adrenal masses (incidentaloma) are detectedincidentally by abdominal CT, and other imaging modalities.¹¹C-metomidate showed almost 100% specificity for the identification ofhormonally silent adrenocortical adenoma, when compared with CT(computed tomography) and MRI (magnetic resonance imaging). Both CT andMRI cannot differentiate silent from hormonally active adrenocorticaladenoma (Bergström et al., 2000; Khan et al., 2003). However, the natureof these masses must be identified to exclude adrenocorticaldysfunction, or metastatic or primary adrenal cortical cancer (Abecasiset al. 1985). While ¹¹C-metomidate specifically detects tissue ofadrenocortical origin, it shows no uptake in cysts, lipoma, hematoma ormetastases of other primary tumors (Khan et al., 2003).

Although ¹¹C-metomidate has “ideal” biological characteristics forscintigraphy of the adrenals and tumor derived therefrom, application ofthe radiopharmaceutical is limited to hospitals with a PET facility. ¹¹Cis a cyclotron product and decays with a half-life of 20 min, therefore,¹¹C-metomidate must be synthesized immediately prior to use.

Halogenations, on the other hand, offer sufficient flexibility, time forpreparation and shipment. (Iodine-123 T_(1/2)=13.2 hours; Br-76T_(1/2)=16 hours; F-18 T_(1/2)=1.8 hours). Modification of the esterfunction offers access to labelled R¹ derivatives, which are equallypotent inhibitors (i.e. ¹¹C-MTO, ¹⁸F-FETO). Substitutions in the phenylring with a radiohalogen produced radiolabelled derivatives that arealso disclosed in U.S. Pat. No. 7,189,859.

Enzyme inhibitors, such as metyrapone have been labelled withradioiodine for adrenal scintigraphy, however, these compounds havenever been used for clinical diagnosis (Wieland, 1982; Robien & Zolle,1983). The compounds in accordance with the present invention potentlyand selectively bind to adrenocortical membranes (cytochrome P-450c11).

A comparison of the binding affinities (IC₅₀ values) of some etomidatederivatives with known inhibitors clearly demonstrate the high potencyof (R)-etomidate derivatives (FIG. 1). Hydrolysis of the ester functionresulted in a loss of binding potency.

With the above and other objects in view there is provided, inaccordance with the invention methods for clinical application of1-(1-arylalkyl)-1H-imidazole-5-carboxylate ester derivatives of formula(I) with modified functionality R¹, R², and R³, incorporating aradioactive halogen, wherein the compound is either prepared shortlyprior to administering to the subject, or prepared at least one daybefore the imaging is performed, and stored until needed.

In accordance with another feature of the invention, there is provided amethod for performing adrenal scintigraphy for the diagnosis ofassociated disease, the method comprising: (a) administering to apatient an effective amount of radioactivity of a compound defined inclaim 2 or 3 wherein R¹ is radioactive 2-fluoroethyl; or a compoundwherein R³ is phenyl, substituted with a radioactive halogen of thefollowing formula:

wherein R¹ and R² are each methyl, and X is radioactive iodine andwherein the compound is *I-metomidate (IMTO); or wherein R¹ is ethyl, R²is methyl, and X is radioactive iodine, wherein the compound is*I-etomidate (1-ETO), wherein the radioactive halogen is selected fromthe group consisting of ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ⁷⁶Br, and ¹⁸F; (b)applying a suitable tomographic procedure, i.e., SPECT or PET

In accordance with yet another feature of the invention, there isprovided a method for adrenal scintigraphy for the localization andcharacterization of abnormal adrenocortical function, wherein saidassociated conditions are Cushing's syndrome; primary aldosteronism; andthe incidentally discovered adrenal mass; especially, adenoma;bilateralcortical nodular hyperplasia; adrenocortical carcinoma;hormonally silent adenoma.

In accordance with an added feature of the invention, there is provideda method for functional adrenal scintigraphy and diagnosis of associateddisease, wherein said associated conditions are selected from the groupof conditions presenting with hyperfunctioning adrenal(s),adrenocortical adenoma, and adrenal tumors, said method comprising:

diagnosis of adrenocortical adenoma; bilateralcortical nodularhyperplasia or diagnosis of metastatic or primary adrenocorticalcarcinoma;

detection of residual masses, staging of tumors and follow-up; and

differentiation between tumors not originating from adrenal cortex.

In accordance with yet another feature of the invention, there isprovided a method for functional adrenal scintigraphy and diagnosis ofassociated disease, wherein said associated conditions are selected fromthe group presenting with incidentaloma, or hormonally silent adenoma,wherein the adrenal-derived tumor is not anatomically confined to theadrenal glands.

In accordance with an added feature of the invention, there is provideda method of administering a compound, wherein positron-emissiontomography (PET) is effective in detecting lesions of adrenocorticalorigin, residual masses; facilitating staging of tumors and follow-up.The associated conditions are selected from the group presenting withincidentaloma, adrenocortical adenoma, and adrenal tumors.

In accordance with yet another feature of the invention, there isprovided a method of parenteral application of a compound defined inclaim 5, wherein the radioactive halogen is selected from beta-emittingnuclides (¹³¹I, ⁸²Br) or alpha-emitting astatine (²¹¹At) for the purposeof radionuclide therapy of adrenocortical or extraadrenal malignancy.

FIG. 1 is a table showing the effect of structural changes of etomidatederivatives, and other Inhibitors by the displacement of ¹³¹I-IMTObinding.

Derivatives of etomidate displaced the radioligand ¹³¹I-IMTO with highpotencies, except the (S)-enantiomer, which showed low binding affinity.The different esters (ETO, MTO, and FETO) showed similar potencies. Thefree acid is inactive. Substitution of the phenyl ring with iodine(4-Iodo-MTO) showed a slight effect, nevertheless, ¹³¹I-IMTO performedas a valuable radioligand in the displacement studies. Metyrapone andketoconazole showed considerably lower binding affinities.

The available radiotracers for imaging the adrenal cortex and adrenalcortex-derived tumors are labeled cholesterol derivatives. These include6β-[¹³¹I]-iodomethyl-19-norcholesterol (NP-59) (Basmadjian, 1975) and6β-[⁷⁵Se]-selenomethyl-19-norcholesterol (Scintadren™) (Sakar, 1976).Both NP-59 and Scintadren™ accumulate in the adrenals slowly, withindays, requiring long-lived radionuclides as a label (Iodine-131T_(1/2)=8.04 days; Selen-75 T_(1/2)=120 days). Iodine-131 is alsoemitting beta-radiation, which contributes considerably to the radiationexposure. The diagnostic use of beta-emitters is no longer state of theart.

In view of the drawbacks of above mentioned agents with respect topatient care (high radiation exposure, repeated imaging procedures) theradiolabelled derivatives of etomidate and metomidate have greatlyimproved radionuclide imaging procedures for the detection and follow-upof adrenal disease.

The invention disclosed herein concerns radioactive compounds with highselectivity for adrenocortical tissue, providing metomidate labelledwith a SPECT or PET radionuclide. ¹²³I-metomidate offers optimal imagingcharacteristics with SPECT, ¹⁸F-etomidate with PET. Labeled with abeta-emitting radionuclide (e.g., ¹³¹I), iodoetomidate, resp.,iodometomidate may have potential for treatment of malignancy that showsincreased uptake of the radiotracer. Therapeutic use in patients isbased on high-affinity binding and slow release of said compounds,offering a sufficiently long residence time for delivering a therapeuticradiation dose.

Etomidate is known as a short-acting hypnotic with an adrenostatic sideeffect as a potent inhibitor of cortisol synthesis (Drake et al., 1998).High affinity binding has been demonstrated for the human mitochondrialcytochrome P450 enzymes CYP11B1 and CYP11B2, which catalyze the finalsteps in the biosynthesis of cortisol and aldosterone. Adrenalsuppression has been observed with doses of 0.04 mg/kg (Diago et al.1988), whereas the intravenous induction dose for anesthesia in adultsis reported as 0.3 mg/kg. The high affinity of ETO derivatives asdisplacers of specific radioligand binding serves as a basis of an invitro binding procedure. ¹³¹I-IMTO was used as a radioligand tocharacterize high affinity binding sites on crude membranes preparedfrom whole rat adrenals

Displacement of ¹³¹I-IMTO Binding

Compounds of formula I and derivatives were evaluated as competitiveinhibitors of [¹³¹I]MTO binding. Test compounds were incubated at0.01-100 nM concentrations. Non-specific binding was determined withetomidate (10 μM). The reaction was initiated by the addition of crudeadrenal membranes and was terminated by filtration through Whatman GF/Bfilters (presoaked in buffer), followed by 2×4 mL washings with buffer.The filters containing membranes with bound radioligand were measured ina γ-spectrometer. IC₅₀ values (the molar concentration of compoundnecessary to inhibit binding by 50%) were determined for each testcompound by non-linear, least squares regression analysis, using aniterative curve fitting routine.

The IC₅₀ values for selected derivatives of etomidate and metomidate arepresented in FIG. 1. The ethyl ester (etomidate) shows the highestpotency (IC₅₀=1.1 nM); the methyl ester (metomidate; IC₅₀=3.7 nM) andthe 2-fluoroethyl ester (FETO; IC₅₀=3.0 nM) have similar potencies. Theiodinated derivative, 4-iodo-MTO showed consistently a slightly highervalue (IC₅₀=9.0 nM)). Moreover, it was demonstrated that(R)-configuration of the methyl substituent at the chiral C-atom isessential for binding, (S)-configuration is not tolerated (IC₅₀=492 nM);cleavage of the ester results in deactivation, the free acid (ETO-acid)is inactive (IC₅₀=123 μM); Metyrapone, a known, clinically usedinhibitor, showed micromolar potency (IC₅₀=1.2 μM) when tested in thisassay.

FIG. 2 is a graphic presentation showing the displacement of specific¹³¹I-IMTO binding by ETO (circles), 4-I-ETO (squares), FETO (triangles),and metyrapol (diamonds).

A comparison of IC₅₀ values obtained by the displacement of specificallybound ¹³¹I-IMTO by structurally related compounds offered insight intothe structural requirements for high-affinity binding in vivo.

In Vivo Evaluation of ¹³¹I-IMTO

Method: ¹³¹I-IMTO was used with a radiochemical purity >99% and aspecific activity of 57 GBq/μmol. The radiotracer (0.5-1.1 MBq; 10-20μCi) was injected into the tail vein of rats (female, 180-220 gram).Groups of four rats were sacrificed at specified times up to 24 hourspost injection. The organs were excised and weighed, the radioactivitywas measured at constant geometry using a γ-spectrometer with aNaI(Tl)-crystal. The data were expressed as percent of injected dose(ID) per organ and as percent of ID per gram tissue.

Results: ¹³¹I-IMTO showed high specific uptake in the adrenals ofapproximately 10% ID/g tissue with a radioactivity plateau exceeding 2hours (FIG. 3). The radiotracer is primarily excreted by the kidneys.Renal activity is attributed to ¹³¹I-ETO-acid, which results fromenzymatic cleavage of the methyl ester. The renal activity is increasingup to 4 hours post injection. Based on calculations of thetarget-to-non-target-ratios obtained in rats, the highest contrast forimaging of the adrenals is observed up to one hour post injection (FIG.3). Based on these results, ¹³¹I-IMTO shows a high potential as aradiotracer for functional imaging of adrenal pathology. Thebiodistribution of ¹⁸F-FETO is shown in FIG. 4. In this case no renalaccumulation of free acid is visible, because the free acid is notlabelled.

FIG. 3 shows the distribution of radioactivity in organs afterintravenous injection of ¹³¹I-IMTO in rats (means ±SD; n=4), followed upto 120 minutes post injection, expressed as a percentage of the injecteddose per gram of organ weight.

FIG. 4 shows Target/Non-Target Ratios obtained with ¹³¹I-IMTO in rats

FIG. 5 shows the distribution of radioactivity in organs afterintravenous injection of ¹⁸F-FETO in rats (means±SD; n=3), followed upto 60 minutes post injection, expressed as a percentage of the injecteddose per gram of organ weight.

Clinical Application

Labelled etomidate derivatives have shown excellent characteristics asadrenal imaging agents. Metomidate binds selectively to tissue rich inP-450c11 enzyme activity, which is specifically expressed inadrenocortical tissue and tumors derived therefrom. High affinitybinding to specific receptor sites requires high specific activity ofthe radiotracer, providing an adequate amount of radioactivity in anegligible mass of labelled derivative.

Safe application devoid of any pharmacological effect is dose related,therefore, trace amounts of the radiotracer are injected in a singleintravenous dose. This is accomplished by carrier-free labelling of PETand SPECT radiotracers with high specific activities. Actually, the PETtracer ¹¹C-MTO is generally produced with a specific activity of 60GBq/μmol, the SPECT tracer ¹²³I-IMTO is available with 100 GBq/μmol (2.7Ci/μmol). External scintigraphy thus is performed with a microdose(0.6-5 μg) of the PET or SPECT tracer avoiding a hypnotic effect as wellas cortisol suppression (SF=>2.800).

The acute toxicity of 4-iodometomidate was determined in mice. Nodose-related mortality was observed by applying 2 microgram/kg bodyweight during a period of 14 days in mice (5 males and 5 females). Miceshowed normal food intake and gained weight. None of the organs showedany pathological changes. The calculated dose would correspond to a100-fold dose of ¹²³I-IMTO used in man.

Mutagenicity: The bacteriological test (Ames Test) using the same dosage(100 microgram/plate), showed no signs of a mutagenic effect.

Radiation exposure: The effective (whole body) dose was calculated as0.0259 mSv/MBq. The effective dose in adults (70 kg) resulting from 185MBq (5 mCi) of intravenously injected ¹²³I-IMTO for adrenal scintigraphyis approximately 4.8 mSv.

Patient dose: 185-220 MBq (5-6 mCi) of the SPECT radiotracer 370 MBq (10mCi) of the PET radiotracer, injected as a bolus.

After the intravenous injection of the radiotracer, the scanningsequence is started. 14 frames are acquired during a total examinationtime of 45 minutes. No blood samples need to be taken. The study isevaluated with respect to tracer uptake in the adrenal lesion, in normaladrenals and in the liver. The imaging results are related to findingsat surgery and biochemical screening.

Contrast for Imaging

Based on calculations of the target-to-non-target concentration ratiosthe highest contrast for imaging of the adrenals is observed up to 2hours post injection (FIG. 3).

Advanced imaging techniques, i.e., ¹²³I-IMTO-SPECT or ¹⁸F-FETO-PET aswell as SPECT/CT and PET/CT offer considerable advantages for thedetection and differentiation of adrenal pathology:

-   -   Identification of hormonally silent adrenocortical adenoma        (avoiding biopsy and biochemical screening);    -   Define nature of incidentaloma to exclude silent adrenal        cortical or medullary dysfunction;    -   Detection of a primary adrenal cortical tumor/exclusion of any        other origin;    -   Differential diagnosis of primary adrenocortical tumor and        metastasis of other origin;    -   Discrimination between tumor originating from adrenal cortex and        from adrenal medulla;    -   Discriminate between benign and malignant adrenocortical tumor        to exclude adrenocortical dysfunction; metastatic or primary        cortical cancer;    -   Visualize metastases or recurrence of adrenocortical cancer.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

FIG. 1: Displacement of ¹³¹I-IMTO binding by etomidate derivatives andother inhibitors (IC₅₀-values).

FIG. 2: Displacement of specific ¹³¹I-IMTO binding by ETO (circles),4-I-ETO (squares), FETO (triangles), and metyrapol (diamonds) atincreasing inhibitor concentrations.

FIG. 3: Distribution of radioactivity in organs up to 120 minutes afterintravenous injection of ¹³¹I-IMTO in rats (means±SD; n=4), expressed asa percentage of the injected dose per gram of organ weight.

FIG. 4: Target/Non-Target Ratios obtained with ¹³¹I-Iodometomidate(IMTO) in rats.

FIG. 5: Distribution of radioactivity in organs up to 60 minutes afterintravenous injection of ¹⁸F-FETO in rats (means±SD; n=3), expressed asa percentage of the injected dose per gram of organ weight.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a compound of the general formula I:

whereinR¹ is linear or branched C₁-C₄ alkyl, and is optionally substituted witha halogen selected from the groups consisting of F, Cl, I or Br;R² denotes an alkyl group containing 1 or 2 carbon atoms; andR³ is phenyl, optionally substituted with a halogen;

As used herein, the expression “alkyl,” includes methyl and ethylgroups, and linear or branched propyl groups. Particular alkyl groupsare methyl, ethyl, 2-fluoroethyl, n-propyl, and 2-propyl.

The term “halogen” as used herein, includes iodine, bromine, chlorine,fluorine, and astatine.

The substituent R¹ on the carboxylic ester group may be transformed toother substituents encompassed by the definition of R¹ by suitablereactions known in the art for the modification of carboxylic acidfunctions, i.e., by hydrolysis and esterification and/ortransesterification. The starting materials for the preparation of thenovel compounds of formula (I) are known or they have been obtained byenantioselective synthesis disclosed previously.

Particularly preferred novel compounds in accordance with the presentinvention are those ester compounds wherein R¹ is alkyl substituted witha halogen, preferably with a radioactive halogen.

The compound of formula (I) in accordance with the present invention issuitably a radiolabelled derivative of formula IA:

whereinR¹ is linear or branched C₁-C₄ alkyl, and is optionally substituted withan alpha-halogen; said halogen being selected from fluorine, preferablyradioactive fluorine;R² denotes an alkyl group containing 1 or 2 carbon atoms.

Preferred are compounds of formula IA, wherein R¹ is 2-fluoroethyl andsaid halogen is ¹⁸F.

The compound of formula (I) in accordance with the present invention issuitably a compound wherein phenyl is substituted with a halogen offormula IB:

whereinR¹ is linear or branched C₁-C₄ alkyl, and is optionally substituted witha halogen selected from the groups consisting of F, Cl, I or Br;R² denotes an alkyl group containing 1 or 2 carbon atoms; andR³ is phenyl, substituted with a radioactive halogen, selected from thegroup consisting of, ¹²³I, ¹²⁴I, ¹²⁵I ¹³¹I, ⁷⁶Br, ⁸²Br, ²¹¹At, or ¹⁸F;

Preferred are compounds of formula IB, wherein said halogen is ¹²³I,¹²⁴I, ¹³¹I, or ¹⁸F.

The present invention is described below in more detail in connectionwith the synthesis of an R¹ derived labelled ester (R)-2-¹⁸F-fluoroethyl1-(1-phenylethyl)-1H-imidazole-5-carboxylate and the R³ derivedradiotracer(R)-1-[1-(4-¹³¹I-iodo-phenyl)ethyl]-1H-imidazole-5-carboxylic acidmethyl ester (¹³¹I-IMTO). Examples are given merely for illustrativepurposes and shall in no way be understood as a limitation of the scopeof the present invention which is given by the patent claims.

EXAMPLES

The substituent R¹ on the carboxylic ester group may be transformed toother substituents defined as R¹ by suitable reactions known in the artfor the modification of carboxylic acid functions. However, introducinga positron emitter requires special techniques for the conversion of theradionuclide into a reactive alcohol for subsequent esterification,generally performed on-line using closed synthesis modules.

Synthesis of Modified Esters

Transesterification of commercially available etomidate at ambienttemperature in dry MeOH, n-propanol, or 2-propanol in the presence ofthe corresponding sodium alkoxide yielded metomidate, and the n-propyland 2-propyl esters, respectively.

Example 1 Synthesis of (R)-2-fluoroethyl1-(1-phenylethyl)-1H-imidazole-5-carboxylate

A solution of DtBAD (0.128 g, 0.554 mmol) in dry toluene (2 mL) wasadded to a stirred mixture of Ph₃P (0.145 g, 0.554 mmol), methyl1H-imidazole-5-carboxylate (0.100 g, 0.462 mmol) and 2-fluoroethanol (44mg, 0.040 mL, 0.681 mmol, handle with care!) in dry toluene (2 mL) underan atmosphere of argon. After 18 h, water (two drops) was added and themixture was concentrated under reduced pressure to give a residue whichwas purified by flash chromatography (first column: 60 g of silica gel,hexane/Et₂O/iPr₂NH 5/10/1, R_(f) 0.25, 98 mg of mixture of 2-fluoroethylester and hydrazo ester; second flash chromatography: 40 g silica gel,Et₂O as eluent, R_(f) 0.30) to give the product (38 mg, 31%) as acrystalline solid, mp 51° C. (hexane); [α]²⁰ _(D)=+106.29 (c 0.72,acetone). Anal. (C₁₄H₁₅FN₂O₂) C, H, N.

Radiosynthesis of (R)-2-¹⁸F-fluoroethyl1-(1-phenylethyl)-1H-imidazole-5-carboxylate (¹⁸F-FETO)

Synthesis is based on the nucleophilic radiofluorination withno-carrier-added ¹⁸F-fluoride after kryptofix 2.2.2.-activatednucleophilic substitution of 1,2-dibromoethane in acetonitrile to yield2-¹⁸F-fluoroethyl bromide for ¹⁸F-fluoroethylation of(R)-1-(1-phenylethyl)-1H-imidazole-5-carboxylic acid as thetetrabutylammonium salt to yield the labeled fluoroethyl derivative. Theradioligand is produced with a specific activity of approx. 40 GBq/μmol(1.1 Ci/μmol).

The substituent R³ is labelled by oxidative destannylation of especiallysynthesized precursors, which facilitate rapid labelling under mildreaction conditions. Therefore, 4-iodo-metomidate or 4-iodo-etomidate,respectively, is converted to the 4-trimethylstannyl derivative to serveas a precursor for labelling metomidate and etomidate with anyradiohalogen.

Radiosynthesis of 4-¹²³1-iodophenyl-metomidate (¹²³I-IMTO)

Radiohalogenated compounds of formula IB are conveniently prepared byreacting a stannylated precursor with radiohalogen (Iodine-123;iodine-131; bromine-76 and others) in the presence of an oxidizingagent, at room temperature. The radioligand ¹³¹I-IMTO is produced with aspecific activity of >50 GBq/μmol, resp. >1.35 Ci/μmol.

Substitution with a radiohalogen in the phenyl ring offers access todiagnostic as well as therapeutic MTO-derivatives. Radionuclides fortherapy are beta- and alpha-emitting halogens, e.g., ¹³¹I, ⁸²Br, and²¹¹At.

While the invention has been described in its preferred form orembodiment with some degree of particularity, it is understood that thisdescription has been given only by way of example and that numerouschanges in the details of synthesis, fabrication, and use, including thecombination and arrangement of parts, may be made without departing fromthe spirit and scope of the invention.

LITERATURE RELATED TO THE PRIOR ART

-   Basmadjian G P, Hetzel K R, Ice R D, Beierwaltes W H (1975)    Synthesis of a new adrenal cortex imaging agent    6□-[¹³¹I]-iodomethyl-19-norcholest-5(10)en-3□-ol (NP-59). J.    Labelled Compd. & Radiopharm. XI: 427-434.-   Sakar S D, Ice R D, Beierwaltes W H, Gill S P, Balanchandran S,    Basmadjian G P (1976) Selenium-75-19-selenocholesterol—a new adrenal    scanning agent with high concentration in the adrenal cortex. J Nucl    Med 17: 212-217.-   W. H. Beierwaltes, D. M. Wieland, R. D. Ice, J. E. Seabold, S. D.    Sarkar, S. P. Gill, and S. T. Mosley: Localization of radiolabeled    enzyme inhibitors in the adrenal gland. J. Nucl. Med., 17(11),    998-1002, 1976.-   W. H. Beierwaltes, D. M. Wieland, S. T. Mosley, D. P. Swanson, S. D.    Sarkar, J. E. Freitas, J. H Thrall, and K. R. Herwig: Imaging the    adrenal glands with radiolabeled inhibitors of enzymes: concise    communication. J. Nucl. Med., 19(2), 200-203, 1978.-   Wu J. L., Wieland D. M., Beierwaltes W. H., Swanson D. P., Brown L.    E.: Radiolabelled enzyme inhibitors—enhanced localization following    enantiomeric purification. J. Labelled Compd. & Radiopharm., XVI(1),    6-9, 1979.-   Wieland D M: Radiolabeled enzyme inhibitors—Adrenocortical enzymes.    In: Receptor-binding radiotracers, Vol. 1, 127-146, Ed. W. C.    Eckelman, Chemical Rubber Co. Press, Cleveland, Ohio, 1982.-   Robien W. and Zolle I. (1983) Synthesis of radioiodinated    metyrapone—A potential agent for functional imaging of the adrenal    cortex. Int. J. Appl. Radiat. Isot. 34: 907-914.-   Zolle, W. Woloszczuk, and R. Höfer: Synthesis and in vitro    evaluation of metyrapone derivatives as potential inhibitors of    11□-hydroxylase activity. In: Radiopharmaceuticals and labelled    compounds, 337-342, IAEA-CN-45/67, Vienna, 1985.-   Yu, J., Zolle, I., Mertens, J., and Rakias, F.: Synthesis of    2-[¹³¹I]-iodophenyl-metyrapone using Cu(I)-assisted nucleophilic    exchange labelling: Study of the reaction conditions. Nucl. Med. &    Biol. 22(2): 257-262 (1995).-   Vanden Bossche H, Willemsens G, Cools W, Bellens D (1984) Effects of    etomidate on steroid biosynthesis in subcellular fractions of bovine    adrenals. Biochemical Pharmacology: 33(23), 3861-3868.-   Belelli, D.; Lambert, J. J.; Peters, J. A.; Wafford, K.;    Whiting, P. J. (1997) The interaction of the general anesthetic    etomidate with the g-aminobutyric acid type A receptor is influenced    by a single amino acid. Proc. Natl. Acad. Sci. USA 94, 11031-11036.-   Franks, N. P. (2006) Molecular targets underlying general    anaesthesia. Br. J. Pharmacol. 147 Suppl 1, S72-81.-   Engelhardt D (1994) Steroid biosynthesis inhibitors in Cushing's    syndrome. Clin Investig 72: 481-488.-   Weber M M, Lang J, Abedinpour F, Zeilberger K, Adelmann B,    Engelhardt D (1993) Different inhibitory effect of etomidate and    ketoconazole on the human adrenal steroid biosynthesis. Clin.    Invest. 71: 933-938.-   Godefroi, E. F., Janssen, P. A. J., Van der Eycken, C. A. M., Van    Heertum, A. H. M. T., Niemegeers, C. J. E. (1965)    DL-1-(1-Arylalkyl)imidazole-5-caroxylate esters. A novel type of    hypnotic agents. J. Med. Chem. 8: 220-223.-   Synthesis of etomidate U.S. Pat. No. 3,354,173 issued Nov. 21, 1967.    Expired Nov. 1984.

Labelled Metomidate

-   Bergström, M.; Bonasera, T. A.; Lu, L.; Bergström, E.; Backlin, C.;    Juhlin, C.; Långström, B. (1998) In vitro and in vivo primate    evaluation of carbon-11-etomidate and carbon-11-metomidate as    potential tracers for PET imaging of the adrenal cortex and its    tumors. J. Nucl. Med. 39, 982-989.-   Bergström, M.; Juhlin, C.; Bonasera, T. A.; Sundin, A.; Rastad, J.;    Åkerström, G.; Långström, B. (2000) PET imaging of adrenal cortical    tumors with the 11β-hydroxylase tracer ¹¹C-metomidate. J. Nucl. Med.    41, 275-282.-   Wadsak, W., Mitterhauser, M. (2003) Synthesis of [¹⁸F]FETO, a novel    potential 11β-hydroxylase inhibitor. J. Label. Compds. Radiopharm.    46, 379-388.-   Mitterhauser, M.; Wadsak, W.; Wabnegger, L.; Sieghart, W.;    Viernstein, H.; Kletter, K.; Dudczak, R. (2003) In vivo and in vitro    evaluation of ¹⁸F-FETO with respect to the adrenocortical and    GABAergic system in rats. Eur. J. Nucl. Med. & Molec. Imaging 30,    1398-1401.-   Schirbel, A.; Zolle, I.; Hammerschmidt, F.; Berger, M. L.; Schiller,    D.; Kvaternik, H.; Reiners, Chr. (2004) [^(123/131)I]Iodometomidate    as a radioligand for functional diagnosis of adrenal disease:    synthesis, structural requirements and biodistribution. Radiochim.    Acta 92: 297-303.-   Hammerschmidt, F.; Peric Simov, B.; Schmidt, S.; Schneider, S.;    Zolle, I. (2005) Chemoenzymatic synthesis of stannylated metomidate    as a precursor for electrophilic radiohalogenations—regioselective    alkylation of methyl 1H-imidazole-5-carboxylate. Monatshefte Chem.    136, 229-239.-   Wadsak, W.; Mitterhauser, M.; Rendl, G.; Schuetz, M.; Mien, L. K.;    Ettlinger, D. E.; Dudczak, R.; Kletter, K.; Karanikas, G. (2006)    ¹⁸F-FETO for adrenocortical PET imaging: a pilot study in healthy    volunteers. Eur. J. Nucl. Med. Mol. Imaging 33, 669-672.-   Zolle I. M., Berger M. L., Hammerschmidt F., Hahner S., Schirbel A.,    Peric-Simov B. (2008) New selective inhibitors of steroid    11b-hydroxylation in the adrenal cortex—Synthesis and SAR of potent    etomidate analogues. J. Med. Chem. (accepted for publication)

Literature on Incidentalomas

-   Siren J E, Haapiainen R K, Huikuri K T, et al. (1993) Incidentalomas    of the adrenal gland: 36 operated patients and review of literature.    World J. Surg. 17: 634-639.-   Reincke M, Fassnacht M, Väth S, Mora P, Allolio B (1996) Adrenal    incidentalomas: A manifestation of the metabolic syndrome. Endocrine    Research 22(4): 757-761.-   Abecasis M, McLoughlin M J, Lange B, Kudlaw J E (1985) Serendipitous    adrenal masses: Prevalence, significance and management. Am. J.    Surg. 149: 783.-   Herrera M F, Grant C S, van Heerden J A, et al. (1991) Incidentally    discovered adrenal tumors: an institutional perspective. Surgery    110: 1014-1021.-   Kloos R T, Gross M D, Francis I R, Korobkin M, Shapiro B (1995)    Incidentally discovered adrenal masses. Endocrin Rev. 16: 460-484.    Lit. Clinical Experience with ¹¹C-metomidate-   Eriksson, B.; Bergstrom, M.; Sundin, A.; Juhlin, C.; Örlefors, H.;    Öberg, K.; Långström, B. (2002) The role of PET in localization of    neuroendocrine and adrenocortical tumors. Ann. N.Y. Acad. Sci. 970,    159-169.-   Khan, T. S.; Sundin, A.; Juhlin, C.; Långström, B.; Bergström, M.;    Eriksson, B. (2003) ¹¹C-metomidate PET imaging of adrenocortical    cancer. Eur. J. Nucl. Med. & Molec. Imaging 30, 403-410.-   Minn, H.; Salonen, A.; Friberg, J.; Roivainen, A.; Viljanen, T.;    Långsjö, J.; Salmi, J.; Välimäki, M.; Någren, K.; Nuutila, P. (2004)    Imaging of adrenal incidentalomas with PET using ¹¹C-metomidate and    ¹⁸F-FDG. J. Nucl. Med. 45, 972-979.-   Hennings, J.; Lindhe, Ö.; Bergström, M.; Långström, B.; Sundin, A.;    Hellman, P. (2006) ¹¹C-metomidate positron emission tomography of    adrenocortical tumors in correlation with histopathological    findings. J. Clin. Endocrinol. Metab. 91, 1410-1414.    Literature Related to Toxic Effect of Etomidate when Used as a    Hypnotic-   Drake W M, Perry L A, Hinds C J, Lowe D G, Reznek R H, Besser G    M (1998) Emergency and prolonged use of intravenous etomidate to    control hypercortisolemia in a patient with Cushing's syndrome and    peritonitis. J. Clin. Endocrinol. Metab. 83: 3542-3544.-   Ledingham I, Watt I (1983) Influence of sedation on mortality in    critically ill multiple trauma patients. Lancet, Jun. 4, 1270.-   Fellows, I. W.; Bastow, M. D.; Byrne, A. J.; Allison, S. P. (1983)    Adrenocortical suppression in multiply injured patients: a    complication of etomidate treatment. Brit. Med. J. 287: 1835-1837.-   Fellows I. W.; Byrne A. J.; Allison S. P. (1983) Adrenocortical    suppression with etomidate. The Lancet, 54-55.-   Allolio, B., Stuttmann, R., Fischer, H., Leonhard, W.,    Winkelman, W. (1983) Long-term etomidate and adrenocortical    suppression. The Lancet ii: 626.-   Wagner R L, White P F, Kan P B, Rosenthal M H, Feldman D (1984)    Inhibition of adrenal steroidogenesis by the anesthetic    etomidate. N. Engl. J. Med. 310: 1415-1421.-   Diago, M. C.; Amado, J. A.; Otero, M.;    Lopez-Cordovilla, J. J. (1988) Anti-adrenal action of subanaesthetic    dose of etomidate. Anaesthesia 43, 644-645.

Lit. for Fluoroethylation

-   Wester H J, Herz M, Weber W, Heiss P, Senekowitsch-Schmidtke R,    Schwaiger M, Stöcklin G (1999) Synthesis and radiopharmacology of    O-(2-¹⁸F-fluoroethyl)-L-tyrosine for tumor imaging. J. Nucl. Med.    40: 205-212.-   Wester H-J, Willoch F, Tölle TR, Munz F, Herz M, Øye I, Schadrack J,    Schwaiger M, Bartenstein P (2000)    6-O-(2-[¹⁸F]fluoroethyl)-6-O-desmethyldiprenorphine (¹⁸F]DPN):    Synthesis, biologic evaluation, and comparison with [¹¹C]DPN in    humans. J Nucl Med 41: 1279-1286.-   Hamacher, K., Coenen, H. H. (2002) Efficient routine production of    the ¹⁸F-labelled amino acid O-(2-¹⁸F-fluoroethyl)-L-tyrosine. Appl.    Radiat. Isot. 57: 853-856.-   Wadsak, W., Mitterhauser, M., Zolle, I. (2002) Synthesis of    [¹⁸F]FETO, a novel PET-tracer for adrenal scintigraphy. Eur. J.    Nucl. Med. & Molec. Imaging Vol. 29: Suppl. 1, S59 (Abstract).-   Wadsak W, Mitterhauser M (2003) Synthesis of [¹⁸F]FETO, a novel    potential 11b-hydroxylase inhibitor. J. Lab. Compd. Radiopharm. 46:    379-388.-   Mitterhauser, M.; Wadsak, W.; Wabnegger, L.; Sieghart, W.;    Viernstein, H.; Kletter, K.; Dudczak, R. (2003) In vivo and in vitro    evaluation of ¹⁸F-FETO with respect to the adrenocortical and    GABAergic system in rats. Eur. J. Nucl. Med. & Molec. Imaging 30,    1398-1401.

1. A compound of the general formula (I)

wherein R¹ is linear or branched C₁-C₄ alkyl, and is optionallysubstituted with a halogen selected from the groups consisting of F, Cl,I or Br; R² denotes an alkyl group containing 1 or 2 carbon atoms; andR³ is phenyl, optionally substituted with a halogen;
 2. The compound offormula (I), wherein the ester is substituted with halogen, suitably asa radiolabeled ester derivative of formula IA:

wherein R¹ is linear or branched C₁-C₄ alkyl, and is optionallysubstituted with an alpha-halogen; said halogen being radioactive; R²denotes an alkyl group containing 1 or 2 carbon atoms.
 3. The compoundof claim 2, wherein R¹ is radioactive 2-fluoroethyl, R² is methyl and R³is phenyl; wherein the compound is ¹⁸F-etomidate (18F-FETO);
 4. Methodsfor clinical application of 1-(1-arylalkyl)-1H-imidazole-5-carboxylateester derivatives of formula (I) with modified functionality R¹, R², andR³, incorporating a radioactive halogen, wherein the compound is eitherprepared shortly prior to administering to the subject, or prepared atleast one day before the imaging is performed, and stored until needed.5. A method for performing adrenal scintigraphy for the diagnosis ofassociated disease, the method comprising: (a) administering to apatient an effective amount of radioactivity of a compound defined inclaim 2 or 3 wherein R¹ is radioactive 2-fluoroethyl; or a compoundwherein R³ is phenyl, substituted with a radioactive halogen of thefollowing formula:

wherein R¹ and R² are each methyl, and X is radioactive iodine andwherein the compound is *I-metomidate (IMTO); or wherein R¹ is ethyl, R²is methyl, and X is radioactive iodine, wherein the compound is*I-etomidate (1-ETO), wherein the radioactive halogen is selected fromthe group consisting of ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ⁷⁶Br, and ¹⁸F; (b)applying a suitable tomographic procedure, i.e., SPECT or PET.
 6. Amethod for adrenal scintigraphy for the localization andcharacterization of abnormal adrenocortical function, wherein saidassociated conditions are Cushing's syndrome; primary aldosteronism; andthe incidentally discovered adrenal mass; especially, adenoma;bilateralcortical nodular hyperplasia; adrenocortical carcinoma; andhormonally silent adenoma.
 7. A method for functional adrenalscintigraphy and diagnosis of associated disease, wherein saidassociated conditions are selected from the group of conditionspresenting with hyperfunctioning adrenal(s), adrenocortical adenoma, andadrenal tumors, said method comprising: a. diagnosis of adrenocorticaladenoma, bilateralcortical nodular hyperplasia, or diagnosis ofmetastatic or primary adrenocortical carcinoma in patients; b. detectionof residual masses, staging of tumors and follow-up; and c.differentiation between tumors not originating from adrenal cortex.
 8. Amethod for functional adrenal scintigraphy and diagnosis of associateddisease, wherein said associated conditions are selected from the grouppresenting with incidentaloma, or hormonally silent adenoma, wherein theadrenal-derived tumor is not anatomically confined to the adrenalglands.
 9. The method of administering a compound defined in claim 2,wherein positron-emission tomography (PET) is effective in detectinglesions of adrenocortical origin, residual masses; facilitating stagingof tumors and follow-up. The associated conditions are selected from thegroup presenting with incidentaloma, adrenocortical adenoma, and adrenaltumors.
 10. A method of parenteral application of a compound defined inclaim 5, wherein the radioactive halogen is selected from beta-emittingnuclides (¹³¹I, ⁸²Br) or alpha-emitting astatine (²¹¹At) for the purposeof radionuclide therapy of adrenocortical or extraadrenal malignancy.